Project Summary/Abstract G protein coupled receptors (GPCRs) control the majority of cellular signaling; both physiological and pathological. Activated GPCRs induce heterotrimer dissociation, generating active G?GTP and G??. Generated G?? controls effectors in multiple signaling pathways. GPCRs and G proteins based chemokine pathway is crucial for proper cellular functions including migration and growth and is highly implicated diseases including cancer. Cancer cells employ chemokine pathway to hijack growth factor receptor (GFR) signaling, primarily through G??, which has the propensity to control cell adhesion, migration and invasion, facilitating metastasis. Although there are 48 members in the family, G?? is often considered a unitary signaling entity. However, our provocative data show that, the 12 G? types differently govern the PM affinity of G??, regulating the efficacy of G?? signaling in a G? identity dependent manner. In Aim 1, we will establish molecular mechanisms that allow G? subunits to possess their carboxy terminus (CT) identity specific PM affinities. In Aim 2, we will seek mechanisms by which PM affinity of G? subunits control the efficacy of G?? effector activation and regulation of chemokine signaling in triple negative breast cancer (TNBC) cells. In Aim 3 of the proposal, we will examine the role of PM affinity of G? and hence he PM-bound fraction of free G?? in regulation of signaling that govern adhesion, migration and invasion of TNBC cells. This project is designed to train and employ undergraduate and graduate students, and uses innovative subcellular optogenetic methods to control signaling, high resolution confocal as well as total internal reflection fluorescence (TIRF) microscopy to monitor signaling and cell behaviors, novel?unbiased image and data analysis methods to extract data, state- of-the-art genome editing methods to alter the PM affinity of native G? in TNBC cells. The proposed work will provide the first analysis of GPCR-G protein signaling regulation by the G? subtype specific PM affinities of G?? in breast cancer cells. Our goal is to deliver the molecular process that controls G?-PM interactions as a new molecular target governing cancer cell adhesion, migration and proliferation.